Candida albicans translocation through the intestinal epithelial barrier is promoted by fungal zinc acquisition and limited by NFκB-mediated barrier protection

The opportunistic fungal pathogen Candida albicans thrives on human mucosal surfaces as a harmless commensal, but frequently causes infections under certain predisposing conditions. Translocation across the intestinal barrier into the bloodstream by intestine-colonizing C. albicans cells serves as the main source of disseminated candidiasis. However, the host and microbial mechanisms behind this process remain unclear. In this study we identified fungal and host factors specifically involved in infection of intestinal epithelial cells (IECs) using dual-RNA sequencing. Our data suggest that host-cell damage mediated by the peptide toxin candidalysin-encoding gene ECE1 facilitates fungal zinc acquisition. This in turn is crucial for the full virulence potential of C. albicans during infection. IECs in turn exhibit a filamentation- and damage-specific response to C. albicans infection, including NFκB, MAPK, and TNF signaling. NFκB activation by IECs limits candidalysin-mediated host-cell damage and mediates maintenance of the intestinal barrier and cell-cell junctions to further restrict fungal translocation. This is the first study to show that candidalysin-mediated damage is necessary for C. albicans nutrient acquisition during infection and to explain how IECs counteract damage and limit fungal translocation via NFκB-mediated maintenance of the intestinal barrier.


Introduction
The majority of life-threatening invasive Candida infections are caused by C. albicans [1][2][3].The WHO have acknowledged the global threat posed by fungal pathogens and recently published a priority list which included C. albicans in the critical priority group [4].C. albicans normally exists as a commensal member of the mycobiota.Within a healthy host the resident microbiota, epithelial barriers, and the host immune system keep C. albicans commensal and prevent translocation over the intestinal barrier into the blood stream [5][6][7].The fungus can, however, transition to a pathogenic state under certain predisposing conditions [5].In fact, systemic infections arise from endogenous C. albicans populations within the gastrointestinal (GI) tract and require translocation across the intestinal epithelium into the bloodstream [6,[8][9][10].Translocation events occur in patients suffering from a variety of predisposing conditions, such as long-term use of broad-spectrum antibiotics or immunosuppression, and the resulting infections are challenging to diagnose, difficult to treat effectively, and associated with high mortality rates [2].However, the specific fungal factors and host mechanisms that contribute to fungal translocation still remain largely undefined.
In an in vitro model of cultured intestinal epithelial cells, C. albicans-mediated damage due to the hypha-associated fungal cytolytic toxin candidalysin was described as a major mechanism of fungal translocation via a transcellular route [11].In line with this, fungal genes that are necessary for hyphal growth or delivery of candidalysin were needed for the full translocation capacity [11,12].Nevertheless, low-level translocation was possible in strains lacking candidalysin, suggesting that further, yet uncharacterized factors and mechanisms contribute to translocation.
The host response to C. albicans infection has been extensively described in oral epithelial cells (OEC).C. albicans infection activates NFκB and c-Jun-based MAPK signaling pathways in OECs independent of fungal morphology [13].A second MAPK signaling phase involving MKP1 and c-Fos is triggered by the activation of epidermal growth factor receptor (EGFR) by candidalysin-mediated host-cell damage [13][14][15].This innate immune response results in the production of cytokines like IL-1β or IL-8 [15,16].Independent of NFκB and MAPK signaling, C. albicans also induces PI3K/Akt signaling during infection of OECs [17].The response of IECs to C. albicans infection is less well characterized.IECs respond via MAPK and TNF signaling as well as activation of NFκB which protects from fungal-mediated host-cell damage [18].Dual-species RNA sequencing has proven to be a powerful tool for identifying the time-resolved infection-specific activities of C. albicans and host cells during their interaction [19][20][21][22].
In this study, we therefore used dual-species transcriptional profiling to investigate the molecular dynamics upon interaction of C. albicans with IECs and to determine which fungal and epithelial processes contribute to IEC damage and fungal translocation.
We found that the candidalysin-encoding gene ECE1 is required for zinc acquisition during invasion of host cells.This indicates that C. albicans-induced host-cell damage supports the acquisition of host micronutrients, in this case zinc, during infection.We also show that ECE1-dependent host damage and subsequent fungal translocation are limited by NFκB-

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation mediated maintenance of the epithelial barrier.Consequently, NFκB activation by IECs limits the pathogenic potential of C. albicans and helps to protect epithelial barrier integrity.

Transcriptional adaptation of C. albicans to IEC infection includes dynamic metabolic shifts and indicates scavenging of host zinc
To characterize the interaction of C. albicans with intestinal epithelial cells (IECs) from early fungal adhesion to initial invasion (up to 6 h) and later translocation and damage phases (12-24 h), we conducted dual-species RNA sequencing of C. albicans-infected IECs over a 24 h time course.Intestinal epithelial cells (C2BBe1) were seeded and differentiated for 12 d after reaching confluency to form a polarized cell layer [23].Fungal and host RNA was isolated from C. albicans and IECs alone as well as from co-incubated samples.Samples were taken before infection (0 h), at 45 min (0.75 h), 3 h, 6 h, 12 h, and 24 h.C. albicans showed a clear time-dependent transcriptional response with distinct clusters for each time point (Fig 1A).C. albicans samples cultured with and without IECs showed similar transcriptional responses and clustered together, indicating the fungal response in this experimental model was largely independent of the presence of host cells (Fig 1A).
To explore the transcriptional changes in C. albicans that are specific for the presence of IECs, we identified infection-specific differentially expressed genes (DEGs).For that, we compared fungal transcript levels in the presence vs absence of IECs at each respective time point (S1 Table ).Using these DEGs, we performed functional enrichment analysis of gene ontology terms (GO; biological process category; S2 Table and Fig 1B) to identify the molecular patterns of infection.
We first looked at stress-or virulence-related functional categories that were enriched upon infection.In the earliest stages of infection, at 45 min, the enrichment analysis detected the term "response to chemical" which contained the stress-related catalase-encoding gene, CAT1, and the thioredoxin-encoding gene, TRR1 (S2 Table and Fig 1B).Decreased mRNA levels of these genes in infected samples compared to medium-only controls (S1 Table ) suggests that C. albicans does not experience significant stress upon early contact to IECs.In late infection stages, however, the category "detoxification" was significantly overrepresented among infection-specific DEGs with increased transcript levels during infection (Fig 1B).These included many genes encoding predicted membrane transporters like the putative ABC transporter, CDR4, and the putative MFS transporter, QDR3 (S2 Table ).This indicates increased stress related to the host or to host-cell damage.
The majority of enriched GO terms did not contain classical virulence-associated categories and did not include genes related to fungal filamentation or virulence.Only few enriched terms, including some related to lipid metabolism and cell adhesion, fall into these categories.Genes involved in lipid metabolism were significantly overrepresented at 6 h among the infection-specific DEGs (Fig 1B).These included many genes needed for ergosterol biosynthesis, which showed increased expression during infection of IECs (S1 and S2 Tables).As ergosterol biosynthesis is important for filamentation, this increased expression may indicate infectionspecific surface changes in invading hyphae [24].Enrichment of the category "cell adhesion" at 12 h similarly points to infection-specific surface changes (Fig 1B).This category includes DEGs coding for adhesins, cell surface proteins and cell wall integrity-related genes, such as EAP1, ALS1, PRA1, HWP1, XOG1, MNT2 and PMT1 (S1 and S2 Tables).
Most infection-specific changes were, however, due to metabolic adaptations of C. albicans.At time points of initial hypha formation prior to substantial host cell invasion (3 h) but also during host cell invasion and damage (12 h and 24 h) [11], the enrichment analysis revealed

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation adaptations in the central fungal carbon metabolism in categories for carbohydrate transmembrane transport, the glyoxylate cycle, and monocarboxylic acid metabolism were enriched at 3 h (Fig 1B).These categories cover glucose transporter genes like HGT2 and HGT12, which are known to be repressed by high glucose levels [25], and genes involved in utilization of non-glucose carbon sources via the glyoxylate cycle and fatty acid beta oxidation, like ICL1, MLS1, FOX2, and PEX5 (S2 Table ).Lower transcript levels of these genes during infection (S1 Table ) indicates that the presence of IECs provides glucose to infecting C. albicans cells even before substantial fungal invasion.In addition, there was an infection-specific increase in translational activity at 3 h, with higher transcript levels of genes within the categories for ribonucleoprotein biogenesis and snRNA metabolic processes (Fig 1B).This indicates infection-specific re-organization of central cellular processes at an early time point.
At 12 h, genes falling under the term "pyruvate metabolic process" were also significantly enriched, including genes relevant for glycolysis (ENO1, CDC19, TDH3, and FBA2) and for   ).The lower transcript levels of these genes points to reduced glucose availability during these later stages of IEC infection (Fig 1B and S1 Table).Finally, at 24 h, the term "monosaccharide catabolism" was overrepresented among the infection-specific DEGs.This suggests that nonglucose C6 carbon sources are available specifically during the host-cell damage phase of infection (Fig 1B).These genes included some involved in galactose metabolism (GAL1, 7,10) and glycolysis (PFK1, 2) which were more highly expressed in the presence of IECs (S1 and S2 Tables).Translation-related processes were again enriched during infection at the 24 h time point (Fig 1B).Potentially, this reflects an adaptation to the more complex environment when C. albicans gains access to the host cell content.
Taken together, our data indicate that C. albicans dynamically adapts to the high availability of its preferred carbon source glucose at 3 h, to glucose limitation at 12 h, and the availability of alternative carbon sources upon host-cell damage at 24 h.
Importantly, not only did our analysis pick up infection-specific changes in macronutrient metabolism, but also adaptation to micronutrient availability.At 12 h, the term "zinc ion transport" was significantly overrepresented among the infection-specific DEGs (Fig 1B).This term contained the zinc transporter genes ZRT101, ZRT2, ZRT3, and ZRC1, which all had significantly reduced transcript levels during IEC infection.The zincophore gene PRA1 and the zinc homeostasis regulator gene ZAP1 (CSR1) showed the same pattern (Fig 1C) [26].While mRNA levels of these zinc-related genes increased over time in both infecting C. albicans and C. albicans in medium only, their levels were higher in the absence of IECs, especially at later stages of infection (S1 Fig) .The presence of their substrates strongly down-regulates the transcription of genes involved in micronutrient acquisition, like those for zinc [27][28][29].Together, our data therefore indicate a stronger starvation for zinc when C. albicans is grown in the absence of IECs.In contrast, other metal-related terms were not overrepresented except for copper ion transport at initial infection stages (45 min) (Fig 1B).In fact, increasing transcript levels of iron-metabolic genes, like the high-affinity iron permease genes FTR1 and FTR2, in both infection and control conditions indicated a general iron starvation response at later time points.Unlike for zinc, this was not rescued by the presence of IECs (S1 Table ).
Overall, our transcriptome analysis indicates that C. albicans adapts its metabolism and surface to the dynamic environmental changes during experimental IEC infection, including acquisition of zinc from the host cells.

Zinc acquisition during infection of IECs is ECE1-dependent
Zinc ion transport was a significantly overrepresented category among infection-specific DEGs during the later stages of invasion and host-cell damage (Fig 1B).This was not the case for genes involved in the transport of other metals or micronutrients, so we hypothesized that zinc may be of particular importance.To determine whether zinc acquisition plays a role during C. albicans infection of IECs, we utilized gene deletion mutants for the zinc importer genes ZRT101 and ZRT2, the zincophore gene PRA1, and the intracellular zinc transporter gene ZRC1.Zrt101, Zrt2, and Pra1 are involved in zinc acquisition and are upregulated by low zinc [27,28]; in contrast, Zrc1 is involved in zinc detoxification, although its transcriptional regulation has not been investigated.Loss of ZRT101, ZRT2, and PRA1 had no impact on adhesion to, invasion of, or translocation through IECs by zinc-pre-starved C. albicans cells (Fig 2A -2C).The strain lacking ZRC1, however, showed decreased invasion and strongly impaired translocation (Fig 2B and 2C).The dramatic decrease in translocation ability (~10-fold) is unlikely to be solely due to the decreased hypha formation (~1.5-fold) of this strain (S2 Fig) .In contrast to these results, we found that loss of ZRT101, PRA1, and ZRC1 results in significantly decreased damage potential, and an almost complete absence of host-cell damage after deletion of both ZRT101 and ZRT2 (Fig 2D).The host-cell damage defect of this zinc uptake impaired mutant (zrt101ΔΔ/ zrt2ΔΔ), but not of the zinc detoxification defective mutant (zrc1Δ/Δ) was rescued by addition of 25 μM ZnSO 4 , a zinc concentration known to promote C. albicans growth while not being toxic (Fig 2D) [28].This restoration of host-cell damage is likely due to increased growth and filamentation as assessed by microscopy images showing that without addition of zinc there is little-to-no fungal growth for the zrt101ΔΔ/zrt2ΔΔ strain (Fig 2E).Therefore, (A) Loss of zinc acquisition and storage genes had no effect on the adhesion of C. albicans to C2BBe1:HT29-MTX intestinal epithelial cells after 3 h.(B) Zinc acquisition and storage genes did not significantly affect the percentage of invasive hyphae after 6 h co-incubation with C2BBe1:HT29-MTX cells.The invasion of zrc1Δ/Δ was reduced, though not to a statistically significant degree.(C) Loss of ZRC1 significantly impaired translocation of C. albicans cells through C2BBe1:HT29-MTX intestinal epithelial cells grown on a porous membrane after 24 h.(D) The host-cell damage of the zrt101Δ/Δ, zrc1Δ/Δ, pra1Δ/Δ, and zrt101ΔΔ/zrt2ΔΔ strains was significantly reduced after 24 h of infection.The severe damage impairment could be rescued with exogenous addition of 25 μM ZnSO 4 during infection for zrt101ΔΔ/zrt2ΔΔ (P < 0.0001), but not for zrc1Δ/Δ (P > 0.9999).LDH release was adjusted by subtracting the release from uninfected and untreated host cells.(E) The addition of exogenous zinc during infection of C2BBe1:HT29-MTX cells increases the fungal growth of the zrt101ΔΔ/zrt2ΔΔ strain to a level similar to that of the WT (BWP17) strain.Fungal hyphae (blue) were stained with calclofluor white (scale bar = 100 μm).All values are shown as the mean with standard deviation.Invasion (B) and translocation (C) data were compared using a one-way analysis of variance (ANOVA) and the host-cell damage (D) data were compared using a two-way ANOVA.We have shown that C. albicans experiences more intense late-stage zinc starvation in the absence of host cells-at time points at which damage of the IECs takes place in infected samples (Fig 1C) [11].We therefore hypothesized that C. albicans acquires zinc from host cells during infection of the intestinal epithelium and that host-cell damage enables this zinc acquisition.The fungal peptide toxin candidalysin, encoded by the ECE1 gene, is the main damaging factor of C. albicans [11,14].To further investigate the interplay between host-cell damage and zinc acquisition and the involvement of candidalysin in this process, we compared the transcriptional zinc starvation response of wild-type (WT, BWP17) C. albicans to an ece1Δ/Δ strain.The strain lacking ECE1 showed significantly higher transcript levels of ZRT2, PRA1, and ZRT3 after 24 h of infection of IECs, consistent with more severe zinc starvation (Fig 3B, 3C, 3D and 3F).A similar pattern was seen for ZRT101 mRNA levels, though the difference was not statistically significant (Fig 3A and 3F).When cultured in the absence of host cells, the WT (BWP17) and ece1Δ/Δ strains showed no significant differences in transcript levels of zinc-related genes (
The majority of infection-specific host DEGs only appear after fungal invasion and resulting host-cell damage.In comparison, vaginal epithelial cells show a uniform early response to various fungal species, but the response diverges at the later time points and is connected to damage potential [19].We sought to determine whether the IEC DEGs at later time points reflect a general response to fungi or rather a specific response to C. albicans filamentation and damage.To determine which of the host responses identified were filamentation-or damagespecific, we extended our transcriptome analysis to compare IECs infected with C. albicans WT, non-damaging (ece1Δ/Δ), and non-filamentous and non-damaging (efg1ΔΔ/cph1ΔΔ) strains.The two mutant strains used (ece1Δ/Δ and efg1ΔΔ/cph1ΔΔ) were constructed in different genetic backgrounds, therefore the gene expression data was always compared to the corresponding WT strains (BWP17 and SC5314, respectively) to minimize the effect of strainspecific, genetic differences on the host response [30].The set of genes that were only differentially expressed in response to the efg1ΔΔ/cph1ΔΔ strain was then categorized as a filamentation-specific response.Genes that were differentially expressed in response to both the efg1ΔΔ/ cph1ΔΔ and ece1Δ/Δ strains, were considered to constitute a damage-specific response.The filamentation-specific response of IECs to C. albicans did not consist of many genes, though it

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation included mitochondria-associated genes (COX7B, ND2, CYTB, ND4L) and some encoding heat-shock proteins (HSP90AA1 and HSP90AA6P), all with lower transcript levels in the nonfilamentous mutant as compared to the WT (SC5314) (Fig 4C and S5 Fig).
The damage-specific response of IECs comprises even fewer genes, which in most cases showed lower transcript levels in IECs infected with the ece1Δ/Δ and efg1ΔΔ/cph1ΔΔ mutants than with the respective WT strains.According to these data, IECs respond to C. albicans-mediated damage via upregulation of EGR1 and EGR2, the FOS genes FOSL1 and FOSB as well as the IL-8-encoding gene CXCL8 (Fig 4C and S5 Fig).c-Fos is a major component of the oral epithelial cell (OEC) response to C. albicans filamentation and damage [13,14].Similarly, EGR1 induction as well as IL-8 secretion are known responses to C. albicans infection in OECs [31].
To test how conserved the response of IECs and OECs to C. albicans and fungus-mediated damage are, we performed western blotting for the OEC response components EphA2 and EGFR (receptors), the transcription factor c-Fos, the phosphatase MKP1 and the kinases Akt and p38 [13,16,17,32].We observed an infection-specific increase in phosphorylation of EphA2 and Akt, as well as an infection-specific increase in c-Fos protein levels.c-Fos signals were consistently reduced in the ece1Δ/Δ mutant.While there was also decreased phosphorylation of EphA2 for the ece1Δ/Δ mutant compared to the WT, this was not consistent for all replicates (S6A and S6B Fig) .Thus, we see a partial conservation in the damage response during C. albicans infection of IECs compared to OECs, with ECE1-dependent induction of c-Fos and potentially phosphorylation of EphA2, but no EGFR or MKP1 phosphorylation and no change in the phosphorylation of p38 or Akt in the absence of ECE1 compared to the WT [14][15][16]33,34].
To verify that production of IL-8 in IECs is filamentation-and damage-dependent, we measured IL-8 secretion in response to C. albicans infections.We compared the WT with the nonfilamenting efg1ΔΔ/cph1ΔΔ strain, the non-damaging ece1Δ/Δ strain, and a previously identified strain (cht2Δ/Δ) with increased damage of IECs [11] (Fig 4D).Indeed, the efg1ΔΔ/cph1ΔΔ and ece1Δ/Δ mutants elicited less IL-8 secretion while cht2Δ/Δ induced more IL-8 secretion than their respective WT strains.Overall these data show that while the response of IECs to C. albicans infection is largely independent of hypha formation and fungal-mediated damage, there is a damage-specific aspect via c-Fos induction and IL-8 secretion.

Intestinal epithelial cells limit fungal damage and translocation via NFκB activation
Most of the pathways overrepresented during infection with WT C. albicans were also found during infection with the non-damaging and non-filamentous strains (S2 Table ), again suggesting that the IEC transcriptional response to C. albicans is largely independent of fungal filamentation or damage.Among other signaling pathways that were triggered by the initiation Red indicates that most DEGs showed increased expression during infection, blue indicates most DEGs showed decreased expression, and grey indicates a mix of DEGs with increased and decreased expression within the category.Dots are shown only for significantly enriched categories (overrepresentation analysis, Benjamin-Hochberg adjusted p-value � 0.05, S2 Table ).(C) Significantly differentially expressed genes between ece1Δ/Δ and efg1ΔΔ/cph1ΔΔ C. albicans strains compared to their respective WT strains during infection of IECs.The color scale represents the Log 2 (fold-change) for each gene during infection in the mutant strain compared to the respective WT strain, with red representing higher expression in the mutant strain and blue representing lower expression in the mutant strain.An asterisk indicates the genes with a statistically significant difference (DESeq2 p < 0.05).The mean MRN values are given in S1 Table .(D) Release of IL-8 by C2BBe1 cells infected with C. albicans strains possessing varying damage potentials.The non-damaging efg1ΔΔ/cph1ΔΔ and ece1Δ/Δ strains induced less IL-8 release, though only significantly for the efg1ΔΔ/cph1ΔΔ strains.Conversely, the high-damaging cht2Δ/Δ strains induced significantly more IL-8 release.(E) NFκB activation as measured by the DNA-binding activity of the p65 transcription factor.Infection with all C. albicans strains increased the DNA-binding activity of p65 compared to the uninfected C2BBe1 cells, though not to a statistically significant degree for the efg1ΔΔ/cph1ΔΔ and ece1Δ/Δ strains.All values are shown as the mean with standard deviation.The IL-8 (D) and NFκB activation (E) data were compared using a one-way ANOVA with a post-hoc S ˇida ´k's multiple comparisons test.).Previous research has shown that C. albicansinduced NFκB activation in IECs limits the damage potential during infection [18].We confirmed the C. albicans-induced NFκB activation in IECs and additionally showed that infection with C. albicans strains with high or low damage potential and even with a nonfilamentous mutant elicit similar activation of NFκB by IECs (Fig 4E).This suggests that NFκB activation is a general epithelial response to the presence of C. albicans (Fig 4E).
To determine whether the previously described NFκB-dependent limitation of IEC damage is predominantly host-or fungal-driven, IECs were infected with the non-filamentous efg1ΔΔ/ cph1ΔΔ and non-damaging ece1Δ/Δ strains in the presence and absence of the potent NFκB activation inhibitor quinazoline (QNZ) [18,35].In agreement with previous data, both tested WT strains elicited significantly more host-cell damage when NFκB activation was blocked (Fig 5A) [18].In contrast, there was no statistically significant increase in damage caused by the strains lacking either ECE1 or both EFG1 and CPH1 (Fig 5A).Treatment of IECs with a DMSO vehicle control had no significant effect on either the damage potential or fungal translocation for the WT (BWP17) and ece1Δ/Δ strains (S7A and S7B Fig).
As host-cell damage of IECs is tightly linked to fungal translocation, the translocation rates of C. albicans in the presence of QNZ were also measured [11].As expected from our host-cell damage data, blocking NFκB activation significantly increased the number of translocated fungal CFUs for both WT strains compared to the untreated controls (Fig 5B).The non-filamentous efg1ΔΔ/cph1ΔΔ strain showed no increase in its translocation rate, which was expected given that this strain cannot form hyphae or damage the host cells (Fig 5A and 5B) [11,36].Surprisingly, despite showing no significant increase in host-cell damage upon QNZ-treatment [<50% of untreated WT (BWP17)], the ece1Δ/Δ strain showed a significant increase in translocation comparable to that of the WT (BWP17) under normal infection conditions (Fig 5B).This increased translocation in the absence of increased host-cell damage was confirmed for the ece1Δ/Δ strain with the use of another NFκB inhibitor with a different mode of action that directly inhibits the DNA-binding activity of the p65 subunit [37,38] (S8A and S8B Fig) .These data together indicate that NFκB can limit C. albicans translocation, at least in part, independently of host-cell damage.
We have previously shown that C. albicans translocation across IECs occurs mainly via a transcellular route and is associated with filamentation-and candidalysin-dependent necrotic host-cell damage [11].The increased translocation of the ece1Δ/Δ strain without any increased host-cell damage upon NFκB inhibition suggests that NFκB activation likely limits a non-damaging route for translocation (Fig 5B).To test this hypothesis, the transepithelial electrical resistance (TEER) of IECs was measured with and without QNZ treatment as a metric for barrier integrity.After 24 h, IECs infected with both WT strains showed decreased barrier integrity upon QNZ treatment, though not to a statistically significant degree (Fig 5C ) [WT (SC5314), P = 0.2466; WT (BWP17), P = 0.0824].These results match the increased host-cell damage and translocation data for both strains.There was also a significant decrease in the barrier integrity for IECs infected with both, the efg1ΔΔ/cph1ΔΔ and ece1Δ/Δ strains upon QNZ treatment, again pointing to a host damage-independent effect of NFκB activation on translocation (Fig 5C).This was also confirmed for the WT and ece1Δ/Δ strains using the second NFκB inhibitor (S8A-S8C Fig) .Non-damaging routes of fungal translocation that still reduce barrier integrity can involve induction of host-cell apoptosis or the use of paracellular routes with degradation of cell-cell connections such as tight junctions or adherens junctions [11].As the NFκB signaling pathway can also induce the expression of anti-apoptotic genes and we observed an overrepresentation of infection-specific DEGs for IECs involved in apoptosis at 12 h when fungal-mediated

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation Together, these data suggest that the ability of IECs to limit cellular damage and translocation through NFκB activation is dependent on the filamentation and damage potential of C. albicans itself, and does not rely on increased apoptosis or other forms of programmed cell death.
To finally test whether the decreased barrier integrity during infection with the ece1Δ/Δ strain was associated with increased degradation of cell-cell junction proteins consistent with increased paracellular translocation, epithelial barriers were stained for E-cadherin 24 h after infection with and without QNZ treatment.Micrographs from three biological replicates were then scored by a blinded observer for the consistency of the cell-cell borders and overall organization of the E-cadherin staining.Non-inhibitor treated uninfected and efg1ΔΔ/cph1ΔΔinfected IECs showed a uniform staining of E-cadherin, indicating an intact host cell layer (Fig 5C and 5E).In contrast, untreated cells infected with either WT strain or the ece1Δ/Δ strain showed a weaker staining (Fig 5E).Inhibitor treatment further decreased the E-cadherin signal in IECs infected with either of the two WT strains, as expected from the inhibitordependent increase in damage (Fig 5A).Importantly, the ece1Δ/Δ strain showed less intact host cells and decreased fluorescence upon NFκB activation inhibition, while the host cell layer was largely unchanged for the uninfected and efg1ΔΔ/cph1ΔΔ-infected IECs when treated with QNZ (Fig 5E).These data indicate that NFκB limits paracellular translocation by stabilizing cell-cell junction proteins like E-cadherin.They also suggest that, upon NFκB inhibition, the physical presence of hyphae can break down intercellular barrier function even in the absence of toxin-mediated damage.
To further investigate the effects of NFκB inhibition on cell-cell junction proteins at a molecular level, we measured the junction proteins E-cadherin and claudin-1 by western blotting for infected IECs with and without QNZ treatment (S10A Fig) .Western blotting showed that the total protein levels of E-cadherin and claudin-1 (normalized to GAPDH protein levels) in C. albicans infected IECs were largely unaffected by NFκB inhibition (Figs 5D and S10B Fig) .There was no consistent difference in E-cadherin levels upon inhibitor treatment during infection with the WT (BWP17) or efg1ΔΔ/cph1ΔΔ strains.However, relative E-cadherin levels upon treatment were reduced for IECs infected with the WT (SC5314) and ece1Δ/Δ strains, though not to a statistically significant degree (Fig 5D).The same trend was true for the ece1Δ/Δ and efg1ΔΔ/cph1ΔΔ strains for claudin-1 (S10B Fig) .Though the decrease in both E-cadherin and claudin-1 for the ece1Δ/Δ strain was not significant, it did correlate with the decrease in barrier integrity (Fig 5C ) and is also consistent with the organization of the cell-cell junction proteins (Fig 5E).In summary, these data show that NFκB activation limits ECE1-mediated host-cell damage and independently limits fungal translocation.It does do by maintaining cell-cell junctions and the epithelial barrier integrity in the absence of direct fungal-mediated host-cell damage. of E-cadherin during C. albicans infection with and without QNZ treatment (scale bar = 50 μm).Inhibition of NFκB activation decreased the organization of E-cadherin at IEC borders upon infection with the ece1Δ/Δ and both WT strains.Representative pictures for each strain and treatment condition are shown from 3 biological replicates with median score values in the inset for each strain and condition.All values are shown as the mean with standard deviation.Hostcell damage (A), fungal translocation (B), and barrier integrity (C) data were compared using a one-way ANOVA.Statistical significance for host-cell damage (A) and fungal translocation (B) data was determined with a post-hoc Tukey's multiple comparisons test, while significance for the barrier integrity (C) data was determined using a post-hoc S ˇida ´k's multiple comparisons test.Statistical significance: **, P � 0.01; ****, P � 0.0001.https://doi.org/10.1371/journal.ppat.1012031.g005

Discussion
In this study, we explored fungal and host factors that contribute to C. albicans translocation across intestinal epithelial cell layers, a process which precedes systemic infections.Dual-species RNA sequencing of C. albicans and human cells revealed the contribution of fungal zinc acquisition to host-cell damage.We confirmed a partially conserved damage-specific response of IECs to C. albicans infection and dissected the role of NFκB activation in limiting fungalmediated damage and translocation.
The C. albicans infection-specific transcriptional response was largely time dependent, with most DEGs appearing at the later stages of IEC infection.This was expected as the time points chosen are associated with different stages of epithelial infection: from adhesion, hypha formation, and invasion to host-cell damage and fungal translocation [11].There was no enrichment of known filamentation-or virulence-related terms at time points later than 45 min.This is likely due to the fact that the expression of many C. albicans virulence factors is associated with the yeast-to-hypha transition [40,41].In our experimental setup, there were strong triggers for filamentation in both the infected and medium-only control samples, and hyphae were present at all experimental time points from 3 to 24 h.Transcriptional changes due to the switch from YPD to DMEM medium may account for some similarities in the transcriptional profiles between the infected and control samples at the earlier timepoints.However, the similar pre-culture conditions have been shown previously to play no major role in the gene expression pattern of C. albicans already after 30 min [42].At around 6 h when epithelial invasion occurs, we however observed an infection-specific increase in transcript levels for genes involved in ergosterol biosynthesis.This may be due to subtle physiological changes that support invasion and damage of IECs, as ergosterol biosynthesis is linked to filamentation in C. albicans [43].This is followed by infection-specific changes in the transcript levels of cell adhesion genes at 12 h.These data suggest that the interaction with IECs induces C. albicans hyphae that are physiologically distinct to hyphae grown without any contact to host cells.Taken together, our data along with that of previous studies indicate that while many different environmental conditions trigger morphologically similar hyphae in C. albicans, their precise molecular compositions depend on the growth conditions in which they form [44,45].
The majority of infection-specific changes in the fungal transcriptome were metabolic in nature.Our data show faster glucose consumption during IEC infection, indicated by decreased transcript levels of glycolysis genes after 12 h.This was unsurprising as both fungus and host compete for glucose during infection.In line with this, previous data showed glucose levels were lower during IEC infection by C. albicans after 12 h compared to the fungus grown in medium alone [46].C. albicans further adjusted its metabolism during infection by altering the transcript levels of genes involved in sugar transport and in amino acid metabolism.After 24 h, when a large portion of the epithelial cells was damaged, there were further fungal metabolic adaptations, especially increased mRNA levels of genes involved in glycolysis and galactose metabolism.As transcription of galactose-metabolic GAL genes is induced in the absence of glucose [47], this may suggest that C. albicans gains access to new non-glucose carbon sources which are released due to extensive host-cell damage.
It has been hypothesized that ECE1/candidalysin-mediated cytolysis may be necessary for nutrient acquisition from host cells during fungal infection [48,49].Our study provides evidence in support of this hypothesis by showing an ECE1-dependent alleviation of zinc starvation during epithelial invasion.This suggests that invasion may contribute to zinc acquisition from the host.Zinc is a vital micronutrient for both pathogen and host alike.The availability of zinc for C. albicans within the host is limited during systemic infection and oropharyngeal candidiasis due to nutritional immunity [50][51][52][53].Depending on the host niche, C. albicans can also encounter high and potentially toxic levels of zinc, such as following phagocytosis by immune cells.While C. albicans is limited for zinc during systemic infection, the fungus does not experience severe zinc starvation during infection of IECs [50,51].
Our observations indicate that C. albicans faces a nutritionally complex environment during IEC infection.In general, the transcript levels of the zinc uptake genes ZRT101, ZRT2, and PRA1 were higher in medium alone than during IEC co-culture.This shows that C. albicans experiences less zinc starvation in presence of IECs and suggests that it can access zinc from the host.Deletion of either component of the Pra1/Zrt101 zincophore system significantly reduced the capacity of C. albicans to damage IECs.The zrt2Δ/Δ mutant was unaffected, and the zrt101ΔΔ/zrt2ΔΔ, which is defective in both known zinc uptake pathways, caused no damage.This suggests that C. albicans primarily utilizes the Pra1/Zrt101 system to acquire zinc from IECs, and that the Zrt2 importer can play an important role in the absence of a functional zincophore.In line with this, zinc supplementation of the media fully restored growth and damage potential of zinc uptake-defective strains.
In the absence of exogenous zinc supplementation, the only zinc source for C. albicans is the host epithelium itself.In line with this, preventing host cell cytolysis by ECE1 deletion resulted in a greatly amplified zinc starvation response, which could again be partially alleviated by exogenous zinc supplementation.These results also fit well with clinical manifestations of candidemia.While the human gastrointestinal tract normally contains high levels of zinc in various complexed forms, zinc deficiency is recognized as a significant risk factor for patients in the pediatric intensive care unit, and oral zinc supplementation is recommended as a prophylaxis [54][55][56].Imbalanced zinc homeostasis within the intestine is also associated with microbial dysbiosis and other intestinal diseases that impair the barrier function of the intestinal epithelium, such as inflammatory bowel disease, irritable bowel syndrome and colorectal cancer [57].In our in vitro models of IEC infection, loss of ECE1 did not affect fungal growth per se and thus addition of exogenous zinc was not sufficient to restore normal host-cell damage or fungal translocation despite the partially alleviated starvation response.This could be due to increased sensitivity of our transcriptional analysis in our experimental model or compensatory activities in zinc mobilization of fungal cells that are sufficient to allow for normal growth.Additionally, most fungal material in our models grows atop the epithelial cells and not invasively.Thus, more complex model systems for prolonged intestinal epithelial invasion would be necessary to determine the contribution of Ece1 to nutrient acquisition from the host, especially the physiological relevance of ECE1-mediated zinc acquisition.
Interestingly, zrc1Δ/Δ exhibited the most severe defect in invasive growth and IEC translocation; it also caused much less damage than the WT, which was not reversed upon zinc supplementation.The primary function of Zrc1 is to detoxify fungal cytoplasmic zinc via compartmentalization within vesicular-like structures called zincosomes [28].This, together with the reduced expression of zinc uptake genes by C. albicans on IECs suggests that the fungus requires either Zrt101/Pra1 or Zrt2 to capture zinc from the host, but that once internalized, the zinc detoxification system is crucial for invasion, host-cell damage, and fungal translocation.
The epithelial infection-specific transcriptional response was also dependent on the time point, though to a lesser degree than that of C. albicans.We observed increased transcript levels of mitochondria-associated genes.As this has been shown previously in vaginal epithelial cells as well, it indicates a potentially conserved early epithelial response to fungal infections via mitochondrial signaling [19].The infection-specific host response at later time points was characterized by MAPK, TNF, and NFκB signaling consistent with earlier findings [18].Using our data, we were able to dissect the conserved specificity of this response in a time-resolved manner and to identify additional aspects of the IEC response and its influence on the interaction with C. albicans.At 12 h when host-cell damage and fungal translocation begin, our data show that there is a limited damage-and filamentation-specific transcriptional response to C. albicans infection.We could, however, show that IECs possess a damage-specific response to C. albicans partially conserved with other epithelial cell types [13][14][15][16][17]. Specifically, IECs show increased expression of FOSB, FOSL1, and CXCL8 in the presence of filamentation and candidalysin.Furthermore, IECs show induction of c-Fos and increased phosphorylation of EphA2 and Akt, though this was largely independent of host-cell damage.This indicates that IECs possess a conserved, albeit reduced, damage-specific response to C. albicans infection compared to OECs.This is further exemplified by IL-8 secretion of IECs which correlates with the damage potential of the infecting C. albicans strain, but is much lower overall as compared to oral and vaginal epithelial cells [19,31].
Our data show that the pathways induced in IECs are largely independent of C. albicans host-cell damage and filamentation.Activation of the NFκB signaling pathway in IECs was common for all C. albicans strains tested, and it has already been shown to limit the damage potential of C. albicans [18].We show that this effect on host-cell damage is dependent on the ECE1 gene, as inhibiting NFκB activation had no significant effect on the damage potential of the ece1Δ/Δ strain.Blocking NFκB activation also increased fungal translocation, though this was independent of ECE1, suggesting that NFκB activation limits fungal-mediated damage and fungal translocation via separate mechanisms.We first hypothesized that NFκB activation could limit fungal-mediated damage by inducing expression of anti-apoptosis genes and preventing programmed cell death [39].However, a pan-caspase inhibitor showed no effect on the damage potential of C. albicans and did not counteract the increased host-cell damage when NFκB activation was inhibited.These data suggest that NFκB activation upon C. albicans infection does not prevent apoptosis or other programmed cell death pathways.The specific mechanism by which NFκB signaling limits fungal-mediated damage remains undetermined, but may involve recently described membrane repair mechanisms of epithelial cells [58,59].
We found increased translocation upon inhibition of NFκB activation to be associated with increased breakdown of the epithelial barrier integrity, independent of fungal host-cell damage potential.This contrasts with an earlier study in which NFκB inhibition did not influence IEC barrier breakdown by C. albicans [18].This discrepancy could be attributed to the different experimental conditions between the previous study and ours.While in our study, there were low remaining levels of transepithelial electrical resistance detected after 24 h of C. albicans infection in the absence of an NFκB inhibitor, no transepithelial electrical resistance measurement could be detected at this time point in the previous study [18].This could be due to different differentiation times for the IECs, cell culture media, pore sizes of the transwell membranes, or the MOIs used for infection.Our results rather suggest that NFκB activation may limit fungal translocation via the paracellular route [6].
We found the protective function of NFκB signaling on the barrier integrity during C. albicans infection to be associated with reduced degradation of cell-cell junction proteins.Under normal experimental conditions, C. albicans strains that filament and produce sufficient amounts of candidalysin are likely able to overcome this and translocate following necrotic host-cell death [11].Strains with low-damage potential, like ece1Δ/Δ, are unable to take this route and are thus significantly diminished in their ability to translocate.In this study, we used immortalized intestinal cell lines as a model to dissect the specific cellular mechanisms behind fungal translocation.Future experiments with more complex in vitro model systems that incorporate more physiological factors, like organ-on-chip or intestinal organoids [60][61][62], will help to understand these interactions better.Nevertheless, our findings provide mechanistic insights into how conditions or treatments that alter activation of NFκB could put patients at risk for systemic candidiasis, such as those undergoing treatment with steroids [1,2,39,63].
Host-cell damage and efficient translocation across the intestinal epithelium by C. albicans are dependent on candidalysin, though both processes also require other fungal factors [11,12].Our data connect zinc acquisition from intestinal epithelial cells to fungal growth and host-cell damage during infection, and reveal a limited damage-dependent response of intestinal epithelial cells to C. albicans.Activation of NFκB signaling in intestinal epithelial cells increased the barrier integrity during infection, which limited fungal translocation especially for C. albicans strains with low damage potential.This suggests that host defense mechanisms at the intestinal epithelium limit paracellular translocation, forcing C. albicans towards the damage-mediated transcellular path to overcome the intestinal barrier.

Candida albicans strains and growth conditions
C. albicans strains used in this study are shown in Table 1.The WT strains SC5314 and BWP17+CIp30 are referred to as WT (SC5314) and WT (BWP17), respectively.Strains were routinely cultivated on/in YPD agar/broth (1% yeast extract, 2% peptone, 2% D-glucose with or without 1.5% agar) at 30˚C.Overnight (O/N) cultures were cultured for 16 h in YPD broth at 30˚C with shaking at 180 rpm unless otherwise specified.Cells were then washed twice with phosphate-buffered saline (PBS) and the cell number was adjusted.
For experiments with prior zinc starvation, C. albicans strains were grown for 24 h in YPD liquid medium supplemented with 1 mM ZnSO 4 at 30˚C with shaking at 180 rpm.The OD 600 was then adjusted to 0.5 in SD liquid medium supplemented with 1 mM ZnSO 4 and incubated at 30˚C for 24 h with shaking at 180 rpm.The cells were then washed twice with 1 mM EDTA, washed twice with water, and then diluted to an OD 600 of 0.5 in Zn-free SD liquid medium in acid-washed, plastic flasks.The fungal strains were then incubated for another 24 h at 30˚C with shaking at 180 rpm.

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation ml Holotransferrin (Calbiochem, Merck), and 1% non-essential amino acids (Gibco, Thermo Fisher Scientific) at 37˚C with 5% CO 2 for no longer than 15 passages.C2BBe1 cells were seeded in 6-well plates at a concentration of 5×10 5 cells/well for RNA isolation.C2BBe1 and HT29-MTX cells were seeded in 96-well plates and transwell inserts (polycarbonate membrane with 5 μm pores; Corning) at a 70:30 ratio (C2BBe1:HT29-MTX) and a total concentration of 2×10 4 cells/well or insert for damage and translocation assays, respectively.For adhesion and invasion assays, C2BBe1 and HT29-MTX cells were seeded in 24-well plates with coverslips at a 70:30 ratio (C2BBe1:HT29-MTX) at a total concentration of 1×10 5 cells/well.All well plates and transwell inserts were coated with collagen I (10 μg/ml for 2 h at room temperature; Thermo Fisher Scientific) and maintained for 12 d at 100% confluency for differentiation with regular medium exchange before infection.Just prior to infection with C. albicans, the medium was removed and fresh DMEM without FBS, Holotransferrin, or 1% non-essential amino acids was added to the cells.

RNA isolation
C2BBe1 cells were cultured for 12 d in collagen-coated 6-well plates.To prepare samples for dual-RNA sequencing and qPCR, the differentiated intestinal epithelial cells (IECs) were infected with 2×10 6 Candida cells/well and incubated at 37˚C and 5% CO 2 for 0.75, 3, 6, 12, or 24 h in serum-free DMEM medium.For qPCR samples with added zinc, the fungal and C22Be1 cells were co-incubated in serum-free DMEM with 25 μM ZnSO 4 for 24 h.In parallel, C2BBe1 and C. albicans cells were cultured individually in 6-well plates in serum-free DMEM medium.C. albicans cells from the O/N cultures were collected as uninfected controls for the fungus (0 h C. albicans), and uninfected IECs were harvested as well (0 h IECs).At each respective time point, the medium was removed, unadhered Candida cells were washed away, and 650 μl of RLT buffer (RNeasy Minikit; Qiagen) was added to each well.The plates were frozen with liquid nitrogen and thawed at room temperature (RT).Each well was scraped and the suspensions were centrifuged for 8 min (20,000×g) to pellet the Candida cells.The supernatant was removed and the host RNA was isolated using the RNeasy Minikit (Qiagen) according to the manufacturer's instructions.The fungal RNA was isolated from the pellet using a freeze-thaw method described previously [69].RNA concentrations were measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) and the quality was controlled using a 2100 Bioanalyzer (Agilent Technologies).Fungal RNA used for q-RT-PCR was isolated as described previously with minor modifications [69].Briefly, fungal pellets were resuspended in AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA) with 0.5% SDS and transferred to screw cap tubes with acid-washed glass beads.An equal volume of phenol/chloroform/isoamylalcohol (25:24:1) was added and the fungal cells were lysed using a FastPrep-24 5GMP Biomedicals.AE buffer was added and the samples were centrifuged for 10 min (20,000×g, 4˚C).The aqueous phase was transferred to a PLGheavy tube (QuantaBio), re-extracted with an equal volume of phenol/chloroform/isoamylalcohol, and centrifuged for 5 min (20,000×g, 4˚C).The RNA was then precipitated overnight at -20˚C with pure ethanol and sodium acetate.The RNA concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) and samples were stored at -70˚C.

Fungal gDNA isolation
Differentiated C2BBe1:HT29-MTX cells in 24-well plates were infected with 4×10 5 C. albicans cells and incubated at 37˚C with 5% CO 2 for 24 h in serum-free DMEM with or without 25 μM ZnSO 4 .The medium was removed, the host cells were lysed with 350 μl RLT buffer

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation (RNeasy Minikit: Qiagen), and the fungal cells were spun down.Fungal gDNA was isolated as previously published with the following modifications [70].The fungal pellet was washed with water once.Equal volumes of lysis buffer (2% Triton X-100; 1% SDS; 100 mM NaCl; 10 mM Tris, pH 8; 1 mM EDTA) and phenol:chloroform:isoamylalcohol (25:24:1) were added and the samples were transferred to tubes with acid-washed glass beads.The fungal cells were disrupted using a FastPrep-24 5G (MP Biomedicals).Half the total volume of TE buffer (10 mM Tris; 1 mM EDTA; pH 7.5) was added.Nucleic acid was precipitated from the aqueous phase and resuspended in 400 μl TE buffer following RNase A treatment (10 mg/ml; Sigma) for 30 min at 37˚C.gDNA was precipitated in pure ethanol, washed and resuspended in 25 μl nuclease-free water.

cDNA synthesis and q-RT-PCR
Isolated RNA (1μg) was treated with DNase I (Baseline-ZERO DNase, Lucigen) according to the manufacturer's instructions and then transcribed into complementary DNA using 50μM of oligo (dT) 12-18 primer (Invitrogen), 150 U of Superscript III Reverse Transcriptase (Invitrogen), and 10 U RNase OUT (Invitrogen).The cDNA was then diluted 1:5 and used for q-RT-PCR with the GoTaq qPCR Master Mix (Promega) in a CFX96 thermocycler (BioRad).The expression levels were normalized against the C. albicans ACT1 gene.
For fungal gDNA samples, EFB1 was amplified from 1μl of template with qPCR Master Mix (Promega) in a CFX Opus Real-Time PCR System (BioRad).All primers used are listed in Table 2.The relative gDNA for each sample was calculated relative to the untreated WT (BWP17) for the respective biological replicate.

RNA sequencing and transcriptional analysis
Individual C. albicans and human C2BBe1 samples were combined for dual-RNA sequencing for uninfected isolated samples for time course data.Both C. albicans and Homo sapiens reads were present in infected samples.C2BBe1 reads were present in infection data for mutant vs. WT comparison.
Library preparation and RNA sequencing were carried out at Eurofins Genomics GmbH (Ebersberg, Germany) using the Illumina HiSeq 2500 platform (time course analysis) or

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation Illumina HiSeq 4000 platform (C.albicans mutant to WT comparisons).For library preparation, after poly(A) enrichment, mRNA was fragmented and random-primed cDNA synthesis was performed followed by adaptor ligation and adaptor-specific PCR amplification.Single sequence reads of 50 bp were produced.Preprocessing of raw reads including quality control and gene abundance estimation was done with the GEO2RNaseq pipeline (v0.100.3)[71] in R (version 3.6.3).Quality analysis was done with FastQC (v0.11.5) before and after trimming.Read-quality trimming was done with Trimmomatic (v0.36).Reads were rRNA-filtered using SortMeRNA (v2.1) with a single rRNA database combining all rRNA databases shipped with SortMeRNA.Reference annotation was created by extracting and combining exon features from corresponding annotation files.Reads were mapped against the joined reference genomes of H. sapiens (Ensembl_GRCh38) and C. albicans (C_albicans_SC5314_version_A22) using HiSat2 (v2.1.0,single end mode).Gene abundance estimation was done with featureCounts (v2.0.1) in single-end mode with default parameters.MultiQC version 1.7 was finally used to summarize and assess the quality of the output of FastQC, Trimmomatic, HiSat, featureCounts and SAMtools.The count matrices with gene abundance data without and with median-of-ratios normalization (MRN) were extracted.Raw files are accessible under the Gene Expression Omnibus accession number GSE237496 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE237496) [72].
Differential gene expression was analyzed using GEO2RNaseq.Pairwise tests were performed using four statistical tools (DESeq2 v1.26.0) to report p values and multiple testing corrected p values using the false-discovery rate method q = FDR(p).Mean MRN values were computed per test per group including corresponding log 2 of fold-changes.Gene expression differences were considered significant if they were reported significant by DESeq2 (q � 0.05) and |log 2 (fold-change[MRN based])| � 1.
Functional enrichment analysis was performed using overrepresentation analysis.Gene ontology (Biological process) for C. albicans was performed by parsing data gene lists to the web application of FungiDB for gene annotation and subsequent analysis using default parameters (https://fungidb.org)[73].Enrichment for human reads against KEGG pathways were computed using g:Profiler from within R (package gprofiler2, v0.2.1).Enriched terms were discarded if background size was below 3 or above 1000 genes.A bash script was used to call the Revigo web app for removing redundant terms (reduction factor = 0.5, removal of obsolete terms = yes) [74].KEGG terms were further filtered to exclude coincidental significant hits that were not relevant for the current study (virus-related categories, "Organismal systems" except "Immune system", "Human diseases", "Drug development").Programming code and data necessary to generate plots shown in this manuscript were deposited at Github: https:// github.com/SchSascha/Cal_Translocation.

Quantification of adhesion, invasion, and hyphal length
Differentiated C2BBe1:HT29-MTX cells in 24-well plates with glass coverslips were infected with 1×10 5 C. albicans cells and incubated at 37˚C with 5% CO 2 for 1 h (adhesion) or 6 h (invasion).For adhesion, samples were fixed with 4% formaldehyde and washed twice with PBS.Adherent C. albicans cells were stained with 10 μg/ml Calcofluor white (CFW) (Fluorescent Brightener, Sigma-Aldrich) in 100 mM TRIS-HCl (pH 9.5) for 30 min at RT.After washing with water, samples were mounted on a glass slide with mounting medium (ProLong Gold Antifade, Invitrogen) and imaged.For each replicate, 12 representative images were counted and the mean number of adherent cells in a defined area was calculated from four independent replicates.For quantification of invasion, extracellular C. albicans hyphae were stained with CFW as stated above.The host cells were then permeabilized with 0.5% Triton X-100 for 5

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation min and washed with PBS.Intracellular C. albicans hyphae were first labelled with 20-25 μg/ ml rabbit anti-C.albicans antibody (Acris) for 3 h at 4˚C.After washing with PBS, 4 μg/ml goat anti-rabbit antibody conjugated to AlexaFluor 488 was added and incubated at 37˚C for 1 h.The samples were washed with PBS and mounted as mentioned above.The number of invasive hyphae was determined from 100 hyphae counted per sample.All samples were imaged using an Axio Observer fluorescence microscope (Zeiss).To quantify hyphal length, 2×10 4 C. albicans cells were seeded per well in a 96-well plate in DMEM and incubated for 3 h at 37˚C with 5% CO 2 .Images were taken using a Cell Discoverer 7 microscope (Zeiss) and the hyphal length was measured using Zeiss Zen3.4 (blue edition).

Quantification of host cytotoxicity (LDH assay)
Differentiated C2BBe1:HT29-MTX cells in 96-well plates were infected with 8×10 4 C. albicans cells and incubated at 37˚C with 5% CO 2 for 24 h.Epithelial damage was quantified by release of lactate dehydrogenase (LDH) from IECs.LDH concentrations were measured using a cytotoxicity detection kit (Roche) according to the manufacturer's instructions and LDH from rabbit muscle (Roche) was used to generate a standard curve.The baseline LDH concentration of uninfected and untreated IECs was subtracted and the corrected LDH release is shown as a concentration (ng/ml) present in the supernatant unless otherwise stated.

In vitro translocation assay
Differentiated C2BBe1 cells or a mix of C2BBe1:HT29-MTX cells in transwell inserts were infected with 1×10 5 C. albicans cells and incubated at 37˚C with 5% CO 2 .Supernatant from the upper compartment was removed 24 hpi and used for LDH measurement as described above.The lower compartment was treated with 20 U/ml zymolyase (Amsbio) for 2 h at 37˚C and 5% CO 2 to detach translocated hyphae.The zymolyase-treated hyphae were then plated on YPD agar, incubated at 30˚C for 2 d, and the colony forming units (CFUs) were counted.
For experiments with NFκB inhibition, C2BBe1 cells were treated with DMEM with 2.5 μM 6-Amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) (EVP4593; Sigma-Aldrich) or either 2.5 or 5 μM N-(6-benzoyl-1H-benzo[d]imidazol-2-yl)-2-(1-(thieno [3,2-d]pyrimidin-4-yl)piperidin-4-yl)thiazole-4-carboxamide (SC75741) (MedChemExpress) in the lower compartment for 1 h prior to infection at 37˚C with 5% CO 2 .IECs were then infected as above.After 24 h the trans-epithelial electrical resistance (TEER) was measured using a volt-ohm meter (EVOM2, World Precision Instruments), supernatants from the upper compartment were collected for LDH measurements, the lower compartment was treated as above to measure CFUs, and the transwell membranes were removed for staining.IECs were fixed with Histofix (Roth) for 10 min at 37˚C then washed with PBS.The cells were permeabilized with 0.5% TritonX-100 for 10 min at RT and washed with PBS.Cells were blocked with 2% BSA for 10 min at 37˚C, washed with PBS, and incubated with a primary mouse anti-E-cadherin antibody (10 μg/ml) (BD Biosciences) for 4 h at 4˚C, then washed with 2% BSA.Cells were then stained with a secondary goat anti-mouse antibody conjugated to AlexaFluor 488 (10 μg/ml) (Invitrogen).Samples were washed with PBS, mounted on glass slides, and images with an Axio Observer fluorescence microscope (Zeiss).Samples were prepared from 3 independent biological replicates and 3 images were taken per strain per condition for each replicate.Images were randomized and scored by a blinded observer.Scores ranged from 1 to 5, with a score of 1 representing a sample with consistent, organized staining of E-cadherin at cell-cell borders throughout the sample.A score of 5 was assigned to samples with no visible staining of E-cadherin at cell-cell junctions.For experiments using the pan-caspase inhibitor (Z-VAD), host cells in transwell inserts were treated for 1 h prior to infection with 25 μM of the inhibitor alone or in combination with 2.5 μM of QNZ as described above.
For experiments with added zinc, the number of fungal cells was adjusted in either DMEM or DMEM + 25 μM ZnSO 4 .Differentiated C2BBe1:HT29-MTX cells in transwell inserts were infected as above, with either DMEM or DMEM + 25 μM ZnSO 4 in the transwell insert and DMEM in the lower compartment.After 24 h incubation at 37˚C with 5% CO 2 , the translocated fungal CFUs were plated as described above.

Immunoblotting
For detection of junction proteins with and without NFκB inhibition, C2BBe1 cells were infected as mentioned for the in vitro translocation assay with and without the NFκB inhibitor QNZ and incubated for 24 h at 37˚C with 5% CO 2 .The transwell membranes were then removed, the host cells were scraped off, and spun down for 5 min at 4˚C at 500×g.The pellets were lysed in RIPA lysis buffer (VWR) supplemented with a protease inhibitor mix (Roche) and Benzonase (Millipore) and chilled for 15 min on ice.The samples were spun down for 15 min at 4˚C at 18,000×g.The supernatants were collected and stored at -70˚C.The protein concentrations of total protein extracts were measured by a Lowry protein assay kit (Bio-Rad Laboratories GmbH). 25 μg of each sample was used for a standard 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) at 30 mA and 200 V. Therefore, lysates were supplemented with SDS sample buffer (50 mM Tris/HCl, 2% (v/v) glycerol, 2% (v/v) β-mercaptoethanol, 1.6% (w/ v) SDS, 0.004% (w/v) Serva Blue G-250, pH 6.8) and boiled for 10 min at 95˚C.After SDS-PAGE with subsequent blotting of the samples onto an Amersham Protran 0.45 μm nitrocellulose membrane at 1.8 mA/cm 2 and 25 V, blots were incubated with 5% (w/v) milk dissolved in TBS (50 mM Tris/HCl, 140 mM NaCl, pH 7.2) for 1 h at room temperature.For specific, immunologic labeling, primary antibodies against E-Cadherin (1:1000, MAB1838, R&D Systems), Claudin-1 D5H1D (1:1000, 13255, Cell Signaling Technology), and GAPDH D16H11 (1:1000, 5174, Cell Signaling Technology) were used in 5% (w/v) milk/TBS for 3 h at room temperature or 16-48 h at 4˚C.After 3 times washing in TBS-T (TBS supplemented with 0.05% (v/v) Tween-20), membranes were incubated for 1.5-3 h with IRDye 800CW donkey anti-mouse or donkey anti-rabbit (1:10000, 925-32212 and 925-32213, LI-COR GmbH) as secondary antibodies, respectively.Immunological detections were carried out using an Odyssey M imaging system (LI-COR GmbH).Blots were analyzed by Image Studio Lite Version 5.0 (LI-COR GmbH).
For detection of immune signaling proteins, C2BBe1 cells were seeded in 6-well plates and infected with WT (BWP17) and ece1Δ/Δ C. albicans as described above for RNA isolation experiments.After 6 h of infection, tissue culture plates were placed on ice, the medium was removed, and the cells were washed with ice-cold PBS.Cells were lysed with 120 μL of RIPA buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1 mM EDTA and 5% glycerol) supplemented with protease and phosphatase inhibitors (1:100 dilution) (Sigma Aldrich).Adherent cells were then scraped, transferred into pre-cooled microfuge tubes and incubated on ice for 30 min.Lysates were clarified by centrifugation at 13,300×g at 4˚C for 10 min.The protein extract concentration was measured using a bicinchoninic acid assay (BCA) (Thermo Fisher Scientific) according to the manufacturer's instructions.Proteins were resolved by electrophoresis on 20% SDS-PAGE gels.Following electrophoresis, proteins were transferred onto nitrocellulose membranes (Bio-Rad).Membranes were blocked in 1× Trisbuffered saline (TBS; Severn Biotech) containing 0.001% Tween 20 (Acros Organics) and 5% skimmed milk powder (Sainsbury's).After washing once with TBST, membranes were incubated with primary antibody (Table 3) with gentle agitation overnight at 4˚C.The following

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NFκB-mediated barrier protection and fungal zinc acquisition during intestinal translocation day, membranes were washed 3 times for 5 min with TBST.Membranes were subsequently incubated with rabbit or mouse secondary antibody (Thermo Fisher Scientific) for 1 h at room temperature and then washed 6 times for 5 min with TBST.Finally, the proteins were detected using Immobilon western Chemiluminescent HRP Substrate (Merck Millipore) and developed with an Odyssey Fc Imaging System (LI-COR).Human α-actin was used as a loading control.

Statistical analyses
All experiments were performed with at least three biological replicates.Data were analyzed using Prism 9.4 (GraphPad Software).All data used to generate the graphs is provided as source data (S3 Table ).

Fig 1 .
Fig 1. C. albicans shows transcriptional changes specific to the presence of IECs during infection.(A) Principal component analysis for WT (SC5314) C. albicans RNA sequencing data.Samples clustered together depending on the time point regardless of the presence of IECs (enterocytes).(B) GO terms significantly overrepresented among infection-specific C. albicans DEGs (*positive regulation of filamentous growth of a population of unicellular organisms in response to chemical stimulus; **biological process involved in interspecies interaction between organisms).Gene ratio represents the proportion of genes within each category that were significantly differentially expressed during infection compared to C. albicans cells only.The color scale represents the mean Log 2 (fold-change) of significant DEGs within each category.Red indicates that most DEGs showed increased expression during infection, blue indicates most DEGs showed decreased expression, and grey indicates a mix of DEGs with increased and decreased expression within the category.Dots are only shown for significantly enriched categories (overrepresentation analysis, Benjamin-Hochberg adjusted p-value � 0.05, S2 Table).(C) Gene expression of zinc-related genes comparing C. albicans cell during infection of IECs to those cultured in medium only.The color scale represents the Log 2 (fold-change) for each gene during infection of IECs compared to medium only, with red representing higher expression during infection and blue representing lower expression during infection.Genes for the zinc transporters ZRT101, ZRT2, ZRT3 and ZRC1; the zinc-scavenging protein PRA1; and the transcription factor ZAP1 (CSR1) were less expressed during infection of IECs.An asterisk indicates the time points with a statistically significant difference in gene expression (DESeq2 p < 0.05).The mean MRN values are given in S1 Table.

Fig 2 .
Fig 2. Loss of zinc acquisition genes in C. albicans decreases damage potential by impairing growth during infection of IECs.(A) Loss of zinc acquisition and storage genes had no effect on the adhesion of C. albicans to C2BBe1:HT29-MTX intestinal epithelial cells after 3 h.(B) Zinc acquisition and storage genes did not significantly affect the percentage of invasive hyphae after 6 h co-incubation with C2BBe1:HT29-MTX cells.The invasion of zrc1Δ/Δ was reduced, though not to a statistically significant degree.(C) Loss of ZRC1 significantly impaired translocation of C. albicans cells through C2BBe1:HT29-MTX intestinal epithelial cells grown on a porous membrane after 24 h.(D)The host-cell damage of the zrt101Δ/Δ, zrc1Δ/Δ, pra1Δ/Δ, and zrt101ΔΔ/zrt2ΔΔ strains was significantly reduced after 24 h of infection.The severe damage impairment could be rescued with exogenous addition of 25 μM ZnSO 4 during infection for zrt101ΔΔ/zrt2ΔΔ (P < 0.0001), but not for zrc1Δ/Δ (P > 0.9999).LDH release was adjusted by subtracting the release from uninfected and untreated host cells.(E) The addition of exogenous zinc during infection of C2BBe1:HT29-MTX cells increases the fungal growth of the zrt101ΔΔ/zrt2ΔΔ strain to a level similar to that of the WT (BWP17) strain.Fungal hyphae (blue) were stained with calclofluor white (scale bar = 100 μm).All values are shown as the mean with standard deviation.Invasion (B) and translocation (C) data were compared using a one-way analysis of variance (ANOVA) and the host-cell damage (D) data were compared using a two-way ANOVA.Statistical significance was determined with a post-hoc Dunnett's multiple comparisons test.Statistical significance: *, P � 0.05; ***, P � 0.001; ****, P � 0.0001.
Fig 2. Loss of zinc acquisition genes in C. albicans decreases damage potential by impairing growth during infection of IECs.(A) Loss of zinc acquisition and storage genes had no effect on the adhesion of C. albicans to C2BBe1:HT29-MTX intestinal epithelial cells after 3 h.(B) Zinc acquisition and storage genes did not significantly affect the percentage of invasive hyphae after 6 h co-incubation with C2BBe1:HT29-MTX cells.The invasion of zrc1Δ/Δ was reduced, though not to a statistically significant degree.(C) Loss of ZRC1 significantly impaired translocation of C. albicans cells through C2BBe1:HT29-MTX intestinal epithelial cells grown on a porous membrane after 24 h.(D) The host-cell damage of the zrt101Δ/Δ, zrc1Δ/Δ, pra1Δ/Δ, and zrt101ΔΔ/zrt2ΔΔ strains was significantly reduced after 24 h of infection.The severe damage impairment could be rescued with exogenous addition of 25 μM ZnSO 4 during infection for zrt101ΔΔ/zrt2ΔΔ (P < 0.0001), but not for zrc1Δ/Δ (P > 0.9999).LDH release was adjusted by subtracting the release from uninfected and untreated host cells.(E) The addition of exogenous zinc during infection of C2BBe1:HT29-MTX cells increases the fungal growth of the zrt101ΔΔ/zrt2ΔΔ strain to a level similar to that of the WT (BWP17) strain.Fungal hyphae (blue) were stained with calclofluor white (scale bar = 100 μm).All values are shown as the mean with standard deviation.Invasion (B) and translocation (C) data were compared using a one-way analysis of variance (ANOVA) and the host-cell damage (D) data were compared using a two-way ANOVA.Statistical significance was determined with a post-hoc Dunnett's multiple comparisons test.Statistical significance: *, P � 0.05; ***, P � 0.001; ****, P � 0.0001.https://doi.org/10.1371/journal.ppat.1012031.g002 Fig 3F and S3A-S3E Fig).The addition of exogenous zinc during co-incubation with IECs did not affect the host-cell damage, fungal translocation, or fungal mass of the WT(BWP17) or ece1Δ/Δ strains during infection of IECs (S4A-S4C Fig).The differences in the transcriptional zinc starvation response between the WT (BWP17) and ece1Δ/Δ strains after 24 h were reduced with addition of zinc, though there was still a trend towards increased transcript levels in the ece1Δ/Δ strain (S4D Fig).These data indicate, that ECE1 is necessary for zinc acquisition during interaction with IECs.

Fig 3 .
Fig 3. Loss of ECE1 increases the transcriptional zinc starvation response during infection of IECs.Fold change in gene expression in C. albicans WT (BWP17) and ece1Δ/Δ strains during co-incubation with C2BBe1 cells for (A) ZRT101, (B) ZRT2, (C) PRA1, (D) ZRT3, and (E) ZRC1.Fold changes are calculated as the normalized gene expression at each time point compared to the respective 0 h control samples.(F) Fold change in normalized gene expression of the ece1Δ/Δ strain compared to WT(BWP17) at 24 h in medium only or during infection of IECs.Gene expression was analyzed by q-RT-PCR and is normalized to ACT1 as a housekeeping gene.All values are shown as the mean with standard deviation.All the ratio data from q-RT-PCR experiments were log-transformed before performing a two-tailed, paired t-test.Statistical significance: *, P � 0.05.https://doi.org/10.1371/journal.ppat.1012031.g003 4B) enrichment analysis was performed on the infection-specific IEC DEGs.Similar to C. albicans, there were few significant infection-specific pathways at the 45 min time point (overrepresentation analysis, adjusted p � 0.05; Fig 4B).Components of the JNK and p38 MAPK pathway were induced upon early infection, including the c-Jun N-terminal kinase (JNK) gene MAPK10 as well as the transcription factor AP-1 components FOS, JUN and JUNB.FOS transcript levels remained high during infection at all later time points (S1 and S2 Tables).

Fig 4 .
Fig 4. The transcriptional response of IECs to C. albicans infection is largely damage-and filamentation-independent. (A) Principal component analysis for IEC RNA sequencing data.Samples from IECs (enterocytes) cultured in medium only cluster together across all time points and separately from those co-incubated with C. albicans.(B) KEGG categories significantly overrepresented among infectionspecific DEGs in IECs.Gene ratio represents the proportion of genes within each category that were significantly differentially expressed during infection compared to IECs only.The color scale represents the mean Log 2 (fold-change) of significant DEGs within each category.

Fig 5 .
Fig 5. NFκB activation limits ECE1-dependent damage and paracellular translocation.(A) Inhibition of NFκB activation using the high affinity NFκB activation inhibitor quinazoline (QNZ) increases the damage potential of C. albicans wildtype, but not for the non-filamentous efg1ΔΔ/cph1ΔΔ strain or the non-damaging ece1Δ/Δ strain towards C2BBe1 cells after 24 h.LDH release was adjusted by subtracting the release from uninfected and untreated host cells.(B) Inhibition of NFκB activation increases fungal translocation of C. albicans after 24 h across intestinal epithelial cells independent of damage potential.(C) Blocking NFκB activation significantly increases the breakdown of barrier integrity after 24 h of the intestinal epithelial cell layer for efg1ΔΔ/cph1ΔΔ and ece1Δ/ Δ, with a similar but not significant effect on both WT strains.(D) E-cadherin protein levels normalized to GAPDH and presented relative to levels in untreated C2BBe1 cells.QNZ treatment further increased degradation of E-cadherin during infection with WT (SC5314) and ece1Δ/Δ.(E) Fluorescent labeling